SUMMARY The stability of the proteome is of central importance to the fidelity of all biosynthetic processes, and contributes significantly to the long-term health of the cell and the lifespan of the organism. While the expression of misfolded proteins is intrinsic to protein biogenesis, the accumulation of damaged proteins has become increasingly recognized as a prominent contributor to aging and age-associated disease. We have shown that chronic expression of damaged proteins in response to stress and aging has devastating consequences on protein homeostasis (proteostasis), resulting in a bewildering collection of phenotypes affecting nearly all biological processes. The aims of this grant are to understand the basis of proteostasis dysregulation during aging, and how these events enhance the risk of disease. To address this, we will examine how the expression of disease associated, metastable, aggregation-prone proteins in C. elegans leads to the collapse of the proteostasis machinery, and the role of potent cytoprotective pathways to restore its balance. This will be presented in three aims: 1. The genetics of proteostasis in aging and disease. We will take a systems approach to identify the gene networks that regulate the folding stability of disease associated aggregation-prone proteins. This will be accomplished by genome-wide RNAi screens in C. elegans to identify the proteostasis network for polyQ-expansion proteins (Androgen Receptor, Ataxin-3, Huntingtin, mutant SOD1, A and related amyloidogenic proteins ADan and ABri, and yeast prions (Sup35). Comparative analysis of these screens will provide a genetic basis to identify common and distinct features of each aggregation-prone protein and how the composition of the proteostasis network reflects the folding stability of each protein, 2. The proteomics of proteostasis in aging and disease. Aging and chronic expression of aggregation-prone proteins interfere with proteostasis, which in turn leads to a further amplification of protein damage as other metastable proteins misfold. We propose a combination of cell biological and proteomic approaches to identify the unstable proteome that is at risk in the face of aging and proteotoxic stress, and 3. Cellular mechanisms of proteostasis collapse. Our results suggest that damaged non-native proteins sequester molecular chaperones, which over time, results in the dysregulation of chaperone-dependent cellular processes. As chaperones vary in cellular concentration and among cell and tissue types, we propose that chaperone sequestration is a contributing factor to cellular dysfunction and tissue pathology and suggest an approach to restore the youthful proteostatic state.